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Abstract

Synthesis of the core/shell-structured Fe3O4/Au nanoparticles by trapping Fe3O4 inside hollow Au nanoparticles is described. The produced composite nanoparticles
are strongly magnetic with their surface plasmon resonance peaks in the near infrared
region (wavelength from 700 to 800 nm), combining desirable magnetic and plasmonic
properties into one nanoparticle. They are particularly suitable for in vivo diagnostic
and therapeutic applications. The intact Au surface provides convenient anchorage
sites for attachment of targeting molecules, and the particles can be activated by
both near infrared lights and magnetic fields. As more and more hollow nanoparticles
become available, this synthetic method would find general applications in the fabrication
of core–shell multifunctional nanostructures.

Keywords:

Introduction

Gold nanoparticles (AuNPs) and superparamagnetic iron oxide nanoparticles (SPIONs)
have been subjects of intensive research in the last decade [1,2]. They are generally considered as biocompatible and are of great interest for diagnostic
imaging and therapeutic applications. SPIONs are currently being used as magnetic
resonance imaging contrast agents in clinic. Heating effect of SPIONs in an alternating
magnetic field has been extensively explored for potential hyperthermia treatment
of cancer [3]. Biomedical applications of AuNPs originate from their surface plasmon resonance
(SPR) effect, a strong enhancement of absorption and scattering of light in resonant
with the SPR frequency, which has been utilized for photothermal ablation treatment
and optical imaging. To tune the SPR wavelength to the near infrared (NIR) region
that is commonly regarded as a 'clear window' for deep tissue penetration of light,
various types of Au nanoparticles such as nanorods [4], nanoprisms [5], nanoshells [6,7], and nanocages [8,9] have been developed and investigated.

In recent years, combining SPIONs with Au to form a composite multifunctional nanoparticles
has attracted considerable attention [10-14]. To date, the effort has been mostly limited on coating iron oxide particles with
a thin layer of Au, where the Au shell not only provides convenient anchorage sites
for functionalization of biomolecules through the well-established Au-thiol conjugation
procedure but also protects SPIONs from dissolution and aggregation. However, by such
an approach, it is difficult, if not impossible, to tune the SPR wavelengths to the
NIR region. Reported core/shell particles usually have their SPR in the visible light
range (from 500 to 600 nm), which limits their optical functions for in vivo applications.
Here, we report an approach to construct iron-oxide/Au core/shell nanoparticles by
trapping iron oxide nanoparticles into hollow AuNPs. Such nanoparticles are magnetic
with their SPR peaks in the NIR region (wavelength from 700 to 800 nm).

Experimental Methods

Synthesis of Porous Hollow Au Nanoparticles

Synthesis of porous hollow Au nanoparticles (PHAuNPs) was detailed in the reference
[15]. The key processes are listed here. The synthesis process was conducted inside a
typical three-electrode electrodeposition cell with Ag/AgCl electrode in 3 M NaCl
solution as the reference [Uep = 0.250 V vs. standard hydrogen electrode (SHE)] and a platinum mesh as the counter
electrode. A stack of two anodic alumina filtration membranes (from Whatman Corp.)
with the pore diameter of about 300 nm and thickness of 60 μm were used as templates.
A 700-nm-thick film of Cu was sputter-deposited to block the pores of the bottom membrane,
and this acted as the working electrode for electrodeposition. A commercial Au sulfite
electrodeposition solution (Techni-Gold 25 ES from Technic Inc.) was used as the electrolyte.
The pH of the solution was about 7.0, which was changed to 6.0 by adding 0.4 M Ni
sulfamate solution. A potential of 0.80 V (vs. Ag/AgCl reference) was applied to the
working electrode using a Princeton Applied Research 273A Potentiostat/Galvanostat,
at which hydrogen evolution occurred. As hydrogen molecules diffused into the pores
inside the membranes, hydrogen nanobubbles formed on the inner wall surface of the
pores. These hydrogen nanobubbles served as templates. The high concentration of hydrogen
molecules in the bubble boundary reduced the Au+ complex ion to form Au clusters. Then, these clusters act as a catalyst to trigger
the autocatalytical disproportionation reaction:

3Au1+(insulfitecomplex)→onAusurfaceAu3++2Au

As a result, an Au shell forms around the hydrogen bubble. Metal Au evolves from clusters,
particles to porous networks, forming PHAuNPs, which adhere to the inner wall surface
of the pores inside both (bottom and top) membranes. The PHAuNPs-loaded top membrane
was ready for the next step of loading Fe3O4 nanoparticles into these PHAuNPs after being washed by passing deionized water through
the membrane several times.

Loading Fe3O4 Nanoparticles into PHAuNPs

The formation of Fe3O4 nanoparticles via alkaline precipitation was conducted by following a previously
reported procedure [16]. Briefly, anhydrous 5.2 g FeCl3 (0.032 mol) and 2 g FeCl2 (0.016 mol) were mixed in 25 ml of DI water containing 0.85 ml of 12.1 N HCl under
vigorous stirring. This aqueous solution flew through the PHAuNP-loaded AAO membrane
using a vacuum filtration setup, which guarantees all PHAuNPs were wetted with the
solution. The membrane was removed from filtration setup and immersed into the solution
for additional 30 min. The wet membrane was then transferred into 5 ml of 0.5% NH4OH and was allowed to sit for 20 min. The color change to yellow–orange indicates
the precipitation of iron oxide particles. After the precipitation, the iron oxide
nanoparticles (~10 nm) formed within the membrane were washed away by flowing DI water
through the membrane using the vacuum filtration setup. The Fe3O4/PHAuNPs core/shell nanoparticles were released into water after the dissolution of
membrane using 2 M NaOH solution. The particles were cleaned by several cycles of
dispersion in DI water followed by centrifugation.

Characterization of Fe3O4/PHAuNPs Core/Shell Nanoparticles

Samples for TEM were made by simply dipping a copper grid into the diluted nanoparticle
water suspension. TEM micrographs were taken using a Hitachi H9500 HR-TEM. Absorption
spectra of the particle water suspensions were measured using a Perkin-Elmer Lambda
19 UV/VIS/NIR Spectrometer. Hysteresis loop of dried particle powder was measured
using a vibrating sample magnetometer.

Results and Discussions

Synthesized PHAuNPs feature a sub-25-nm shell with a 50-nm hollow core. The shell
is of porous nature with the pore size about 2–3 nm, as measured in the high-resolution
transmission electron microscopy (HRTEM) image shown in Figure 1a. These nanoscale pores in the shell allow ions (Fe2+ and Fe3+) to diffuse into the hollow space in the core, where precipitation of Fe3O4 takes place upon the addition of OH-. The sizes of precipitated Fe3O4 nanoparticles (5–20 nm) are larger than the pore size, resulting in the trapping
of the iron oxide nanoparticles inside the PHAuNPs (Figure 1b).

Figure 1.a and b HRTEM micrographs of PHAuNPs, showing the hollow core and the porous shell
with pore size about 2–3 nm.

Figure 2 shows TEM analysis before and after loading of iron oxide nanoparticles. After loading,
the hollow core of PHAuNPs is occupied by solid substances. During the precipitation,
Fe3O4 nanoparticles also formed outside of PHAuNPs, but TEM micrographs clearly show that
no small iron oxide nanoparticles were attached to the PHAuNP surface. This is in
agreement with the common notion that iron oxide usually does not stick to the Au
surface [11]. Given the very different sizes of PHAuNPs (~100 nm) and non-trapped Fe3O4 nanoparticles (<20 nm), they can be readily separated using centrifugation.

Figure 2.TEM micrographs of PHAuNPs before a and after b loading iron oxide nanoparticles.

The loading of Fe3O4 to the core of PHAuNPs was confirmed by energy-dispersive X-ray (EDS) analysis of
one single particle and the selected area electron diffraction (SAED) pattern from
three particles. EDS shows the coexistence of Au and Fe in a single particle (Figure
3a, Cu peak is from the TEM grid). The low intensity of Fe may be due to the shield
effect of the thick Au shell. The SAED pattern is a superposition of Au and Fe3O4 lattices (Figure 3b), showing three distinguishable planes of (311), (511), and (731) from Fe3O4. Other Fe3O4 planes overlap with Au planes.

Figure 3.a EDS spectrum of one single particle, showing the coexistence of Au and Fe. b SAED pattern from three particles, showing a superposition of Au and Fe3O4 lattices.

Shown in Figure 4a is the appearance of a bottle of particle water suspension. The cyan color indicates
that the suspension absorbs red light. The absorption spectrum is shown in Figure
4b, which has a broad peak centering at 750 nm. This absorption peak corresponds to
the SPR wavelength. Compared to PHAuNPs before loading iron oxide, the absorption
spectrum shows little change. For core/shell nanoparticles, it is well known that
the SPR wavelength is dependent on the refractive indices of medium, shell and core.
Changing core material usually causes a shift of the SPR wavelength. However, PHAuNPs
have a relatively thick shell (>20 nm). Through a three-dimensional finite difference
time domain (FDTD) simulation (using a commercial software from Lumerical Inc), we
have proved that at this thickness, the red-shifts of SPR peaks are mainly caused
by their surface roughness, and the hollow nature of these particles plays only a
minor role [17]. The simulation results show that SPR peaks for hollow particles are only slightly
red-shifted compared to solid particles with the same outer diameter (100 nm). For
particles with a roughness of 5 nm, SPR peak shifts to longer wavelength (~630 nm).
As the roughness increases to 8 nm which is the average grain size in the shell, a
much greater red-shift (to 720 nm) is observed. This roughness effect is due to the
strong interaction of electric fields from adjacent bumps on the surface, similar
to the plasmonic properties of the aggregates of several nanoparticles. The simulated
results are in good agreement with experimental results. This unique SPR tuning mechanism
makes it possible to maintain the optical properties of PHAuNPs even after the loading
of iron oxide.

Figure 4.The plasmonic and magnetic properties of the Fe3O4-loaded PHAuNPs. a Appearance of a bottle of particle water suspension. The particles can be dragged
toward a permanent magnet. b Absorption spectrum of the particle water suspension, showing a broad peak centering
at 750 nm. c Hysteresis loop of dried particle powder, showing that the suspension consists of
a mixture of superparamagnetic and ferromagnetic nanoparticles.

As shown in Figure 4a, the particles can be dragged toward a permanent magnet, unequivocally indicating
the magnetic characteristics of the Au nanoparticles. Hysteresis loop of dried particle
powder is shown in Figure 4c. Since the Fe3O4 nanoparticles synthesized using the above-mentioned method are normally smaller than
20 nm, we expect to see a typical superparamagnetic behavior: zero remanence, zero
coercivity, and a large saturation field. The small hysteresis shown in the measurement
may reflect the presence of some large Fe3O4 nanoparticles (>30 nm) inside PHAuNPs. Given the size of the hollow space (>50 nm)
and the thickness of the porous shell (25 nm), the inward diffusion of OH- ions may be partially obstructed, resulting in a much slower nucleation rate. As
such, the inside particles could grow large. The measured high saturation field is
in consistence with the superparamagnetic characteristic. This suggests a mixture
of superparamagnetic and ferromagnetic nanoparticles. Ferromagnetic nanoparticles
are usually undesirable for bioapplications because of their agglomeration caused
by magnetic attraction. However, for iron oxide nanoparticles-loaded PHAuNPs, the
thick Au shell can effectively separate them far apart to avoid such magnetic aggregation.

Conclusion

We have shown that the core/shell-structured Fe3O4/Au nanoparticles can be synthesized by trapping Fe3O4 nanoparticles inside hollow Au nanoparticles. Because the resulted composite nanoparticles
combine the desirable magnetic and plasmonic properties into one nanoentity, they
are particularly suitable for in vivo diagnostic and therapeutic applications, where
the Au surface provides anchorage sites for attachment of functional molecules and
the particles can be activated by both NIR light and magnetic field. As more and more
hollow nanoparticles become available, we believe that this synthetic method would
find general applications in the fabrication of core–shell multifunctional nanostructures.

Acknowledgements

This work was supported by the National Science Foundation (ECCS-0901849), the Texas
Higher Education Coordinating Board Norman Hackerman Advanced Research Program, and
the USAMRMC Prostate Cancer Research Program (W81XWH-05-1-0592). We thank the Characterization
Center for Materials and Biology (CCMB) at University of Texas at Arlington for providing
financial and technical support for the electron microscopic characterization of the
nanoparticles.